MiR-126-3p及其靶基因参与强直性脊柱炎发病机制的研究

Excessive proliferation and differentiation of osteoblasts in enthuses result in the ectopic bone formation is the main cause of disability in Ankylosing Spondylitis (AS) patients, but its mechanism is not clear. Our previous study showed that miR-126-3p expressed in peripheral blood mononuclear cells in patients with AS is much lower than control groups.Preliminary bioinformaticsanalysis of its target genes were IRS1 involving in osteogenic differentiation. We proposed to verify IRS1,the target gene of miR-126-3p, by luciferase assay, then culture MC3T3-E1 cells and induce it to osteogenic differentiation.After overexpression and suppression of the miR-126-3p, the changes of IRS1，PI3K，AKT，MAPK and ERK，as well as ALP activity, bone-related markers and mineralization nodules were test. Further, we investigate the differential expression of miR-126-3p in the ectopic bone formation in mouse models; deeply interpret the role of miR-126-3p in initialankylosing and AS development.

肌腱端成骨细胞的过度增殖和分化导致异位骨形成是造成AS致残的主要原因, 但其机制未明确。我们前期研究发现AS患者外周血单个核细胞中miR-126-3p明显低于健康对照组，通过生物信息学预测其靶基因之一是IRS1参与了成骨分化。我们拟通过双荧光素酶报告系统对miR-126-3p靶基因IRS1进行验证，然后培养MC3T3-E1细胞，诱导其向成骨分化，通过过表达miR-126-3p和抑制miR-126-3p表达后IRS1/PI3K/ AKT、IRS1/MAPK/ERK信号通路中的IRS1、PI3K、AKT、MAPK、ERK蛋白表达的变化，以及ALP活性、成骨相关标志物以及矿化结节的影响。最后，我们进一步通过动物实验探索差异表达miR-126-3p对小鼠异位成骨分化的调控，进一步阐明miR-126-3p-IRS1参与AS骨性强直的发生和发展。

经过近3年多的研究，“MiR-126-3p及其靶基因参与强直性脊柱炎发病机制的研究”项目完成了预期计划：. 背景：强直性脊柱炎是一种中轴关节受累为主的慢性炎症性疾病，致残率高。异位骨形成造成的脊柱强直是致残的主要原因。目前发病机制未明。前期MiRNA芯片研究提示miR-126-3p差异表达。因此本研究拟进一步验证及探讨其可能参与强直性脊柱炎发病的机制。. 主要研究内容和结果是：. 1、miR-126-3p在强直性脊柱炎患者中差异表达：通过扩大样本量验证miRNA芯片结果，明确miR-126-3p在强直性脊柱炎患者中的表达情况。结果：与类风湿关节炎组和健康对照组相比，AS患者外周血单核细胞中miR-126-3p明显下调。结论：miR-126-3p在AS患者PBMC中表达明显下降。. 2、miR-126-3p靶基因的预测及验证：利用生物信息学靶基因预测软件预测miR-126-3p的靶基因，并通过双荧光素酶报告基因验证。结果发现：IRS1是miR-126-3p的直接靶基因之一。. 3、miR-126-3p调控成骨细胞分化并参与AS发病机制研究。结果：miR-126-3p可通过抑制IRS1，抑制抑制成骨细胞分化。. 4、初步构建miR-126-3p转基因小鼠并稳定建系。. 项目关键数据和科学意义：进一步验证了miR-126-3p在AS患者中差异表达，实验研究表明其可能参与AS成骨分化异常的机制。这提示miR-126-3p可能是AS致病的miRNA靶点。该miRNA的检测可能为病人疾病诊断的标志物及为新药研发提供理论依据。